Nontuberculous mycobacterium is a gram positive bacillus having acid-fast characteristics classified into genus Mycobacterium (hereinafter, optionally abbreviated simply as M.), and is a kind of acid-fast bacterium other than tuberculosis complex and Mycobacterium leprae. Fifteen to 20% of cases showing positive for sputum smear examination for acid-fast bacterium have been diagnosed to be nontuberculous mycobacterium by subsequent examinations for identification of bacterial strain.
Among nontuberculous mycobacteria, clinically problematic known bacterial strain includes M. avium, Mycobacterium intracellulare (hereinafter, abbreviated as “M. intracellulare”), Mycobacterium kansasii, Mycobacterium marinum, Mycobacterium gordonae, Mycobacterium szulgai, Mycobacterium xenopi, Mycobacterium fortuitum, Mycobacterium chelonei, Mycobacterium abscessus, and so on.
Above all, the commonly noted strains are M. avium and M. intracellulare. Since M. avium and M. intracellulare are closely resemble each other and difficult to distinguish between them, M. intracellulare and M. avium have been referred to collectively as Mycobacterium avium complex (MAC). About 70% of patients with nontuberculous mycobacteria disease are MAC infection, and the second large population is M. kansasii infection accounting 20%. And the rest of 10% are the infection by other bacterial strains.
In general, the nontuberculous mycobacteria have weak toxicity, and they are believed to be harmless to a healthy subject. However, on rare occasions, they may exert infectivity to human. Among them, the MAC is known to cause sometimes aftereffects of tuberculosis (lung infectious disease), or to cause opportunistic infections to a compromised patient such as AIDS patient. Therefore, it is particularly important in the therapy to detect the nontuberculous mycobacteria with rapidity and preciseness.
In addition, in recent years, the incidence of nontuberculous mycobacterium infection demonstrates upward trend, and therefore, development of a method for discriminating tuberculosis bacterium from nontuberculous mycobacteria in a short period of time has been desired strongly. Moreover, from the viewpoint of the fact that the method of detecting/diagnosing M. avium and M. intracellulare by nucleic acid amplification technology has been included in health insurance coverage, its diagnostic significance is obviously great.
In addition, most of the nontuberculous mycobacteria demonstrate resistance against antituberucular agents. Therefore, when a patient is suspected of acid-fast bacterium infection, differential diagnosis whether the disease is tuberculosis or nontuberculous mycobacterium disease is quite important to decide a course of treatment. Further, as a method for treatment of the diseases caused by nontuberculous mycobacteria may vary depending on the individual species of bacterium, the identification of bacterial strain is also quite important. However, nontuberculous mycobacterium diseases do not show any specific clinical symptom. Therefore, it is quite difficult to differentiate tuberculosis from nontuberculous mycobacterium disease by clinical observation and histopathological manifestation, and to specify the species of the nontuberculous mycobacterium. Consequently, the diagnosis whether the disease is tuberculosis or nontuberculous mycobacterium disease has to be determined by bacterial identification of the infected bacterium.
A typical method for identification of bacterium to be carried out for the diagnosis of nontuberculous mycobacterium disease is sputum smear examination. However, by this test, what can be figured out is only whether the pathogenic bacterium is “positive for acid-fast bacterium” or not and whether the pathogenic bacterium is tuberculosis bacterium or nontuberculous mycobacterium cannot be differentiated. Therefore, when the result of the sputum smear examination is positive, bacterial culture examination by isolation culture on a culture medium such as Ogawa's medium has to be carried out to differentiate between tuberculosis bacterium and nontuberculous mycobacterium. And further, by performing additional biochemical examinations, bacterial species of the infected bacterium is identified. However, in general, growth of bacterium belonging to genus Mycobacterium is slow; for example, it takes 3 to 4 weeks only for its isolation culture. And further, it requires additional 2 to 3 weeks to obtain the results of various biochemical tests for the identification of bacterial species. Accordingly, the conventional basic method, in which a diagnostic outcome on whether the disease is tuberculosis or not is obtained by conducting the above described smear examination and a cell culture examination, is a considerably time-consuming method.
On the other hand, in recent years, a technology of detecting bacteria on a genetic level has been developed. For example, a diagnostic technique utilizing the nucleic acid amplification technology such as polymerase chain reaction (PCR) and the like has been studied as a useful means for detecting bacteria. Because of high sensitivity of this method, even if there are only several cells of the bacteria in a sample, the bacteria can be detected. In addition, this method has an advantage that the detection (identification of bacterial species) can be completed in a short time (in 4 days at the longest). However, in the usual PCR method, the number of bacterial count cannot be determined. In addition, in this method, cells are detected regardless of live cells or dead cells. Further, if some bacteria exist in the sample, the determination is made positive regardless of size of the bacterial count. Therefore, by the PCR method, diagnosis on whether the bacteria detected in a patient are infectious or not will be uncertain. Furthermore, the method has a problem such as providing a frequent false positive judgment due to its too high sensitivity.
With respect to the method for detection of M. avium using the PCR method, there is a method for detection of MAC nucleic acid using a multiple primer set of oligonucleotide primers specific for 2 or more of gene regions comprising, for example, MacSequevar gene region, 19 kD protein (MAV 19k) gene region of M. avium, and ribosomal protein s1 gene region of M. intracellulare (Patent Literature 1). However, by this method, discrimination between M. avium and M. intracellulare cannot be achieved. In addition, the Patent Literature 1 has also disclosed an Msqv-Av probe which is a primer capable of detecting only M. avium, and an MAV 19K primer specific for nucleic acid from M. avium. However, even by the detection method using these probe and primer, there also disclosed some cases where M. avium can not be identified clearly as M. avium depending on the type of strain. That is, the specificity of these probe and primer for M. avium is far from satisfactory.
In addition, a method in which PCR is carried out by using a primer capable of amplifying a DNA nucleotide sequence nipping insertion site of the gene insertion sequence IS901, and by determining whether it is avian tuberculosis bacterium (M. avium) or M. intracellulare based on the chain length of obtained primer extension product (Patent Literature 2) has also been known. However, in the PCR using aforementioned primer, the primer extension product can be obtained in either the case where the sample is avian tuberculosis bacterium (M. avium) and the case of M. intracellulare, and therefore, this determination method can not be said as a specific method for M. avium. In addition, the method, whereby the discrimination between both bacterial strains is carried out based on the chain length of the primer extension product is cumbersome; and it is conceivable that different determination may be made by different judge; and in consequence, it cannot be said that the method is a reliable determination method.
Other than the PCR method, there is a determination method through the use of Strand Displacement Amplification Method (SDA method). For example, JP-A-H10-4984 (Patent Literature 3) discloses a method in which 63 nucleotide segment of BCG85-B gene coding a part of α-antigen of mycobacterium is targeted. In this method, using a primer which is capable of amplifying the target sequence in the BCG85-B gene owned by both M. avium and M. intracellulare, nucleic acid amplification reaction is carried out by the SDA method. And then, MAC is detected based on the results. That is, the primer used in aforementioned method is a primer capable of amplifying both M. avium and M. intracellulare. However, in this method, as a matter of course, a primer extension product will be obtained in both cases where either of M. avium or M. intracellulare exists in a sample. Because of that, MAC can be detected by this method; however, it is impossible to detect M. avium specifically. In addition, even when MAC is detected, there can be an instance where false-positive result is provided.
In JP-A-2001-103986 (Patent Literature 4), primers to be used for the detection of MAC, oligonucleotides to be used as a capture probe and a detection probe have been disclosed. However, aforementioned primer can amplify a 48 bp target sequence of dnaJ gene which is owned commonly by both avian tuberculosis bacteria (M. avium) and M. intracellulare. Namely, amplification reaction will take place in both cases where either of M. avium or M. intracellulare is present in a sample. Therefore, if the SDA method is practiced using aforementioned primer, the primer extension product will be detected using the capture probe and detection probe, and based on the results, detection of MAC can be achieved. However, specific detection of M. avium is impossible to achieve without detection of M. intracellulare. 
Beyond that, there is a method of amplification of nucleic acid from M. avium through the use of LAMP (Loop-Mediated Isothermal Amplification) method, and the like. However, in the LAMP method, there are some problems, for example, the nucleotide sequence of amplified DNA cannot be determined; efficient length of DNA to be amplified is limited; and the method provides false-positive result occasionally.
In addition, there exist plural numbers of serotypes such as, for example, serotypes 4 to 6, 8 to 11 and 21 for M. avium, and serotypes 7, 12 to 20 and 25 for M. intracellulare. Therefore, it is difficult to find out a consensus sequence between strains. In consequence, it is difficult to detect all the M. avium having various serotypes by PCR using single primer set. This is the present situation in this field.
As described above, in such the situation, it has been desired to establish a method which enables to detect M. avium specifically and rapidly.
Patent Literature 1: JP-A-H11-69999
Patent Literature 2: JP-B-3111213
Patent Literature 3: JP-A-H10-4984
Patent Literature 4: JP-A-2001-103986
Patent Literature 5: JP-A-2005-204582
Non-Patent Literature 1: F. Poly et al., J. Bacteriology, 2004, 186(14), p. 4781-4795.